Anoxia/ischemia will induce reversible changes in neurons that eventually become irreversible, and the cells die. These changes include alterations in the membrane properties of the neurons as measured by intra- and extracellular recording electrodes. These membrane changes consist of an initial early depolarization followed by a transient hyperpolarization and a subsequent later depolarization that becomes irreversible. The transition from a state of reversible to irreversible cell injury appears to be mediated by a complex chain of events in which calcium and glutamate serve as important links. The research outlined in this proposal will identify the essential steps in this transition in the hope that this understanding will help to point to potential targets for pharmacological intervention and treatment of ischemia-induced cell death. This research will be performed in 5 separate sets of experiments in which the in vitro hippocampal slice will be exposed to anoxic/ischemic conditions; the first 4 sets will use extracellular and intracellular current clamp techniques, and the 5th will use patch clamp techniques. The experiments are as follows: 1. Determination of conditions that result in reproducible irreversible anoxia/ischemia-induced depolarization. 2. Determination of the role of synaptic, glutamatergic innervation in the triggering and maintenance of the irreversible depolarization by lesion and pharmacologic studies. 3. Determination of the role of calcium, as both a charge carrier and as a second messenger, in the establishment and maintenance of the late irreversible depolarization. 4. Determination of the role of second messengers other than calcium in the establishment and maintenance of the late irreversible depolarization. 5. Evaluation of whole-cell and single-channel currents under anoxic/ischemic conditions with patch clamp techniques.